It is proposed to use a newly derived mouse epidermal clone 291, selected both for high efficiency and maintenance of terminal differentiation response to Ca++ in an assay designed to more precisely quantitate chemical carcinogen effects in terminally differentiating epithelial cells, Alteration of the response to calcium is quantitated by counts of colonies which proliferate in 1.4 mM Ca++ relative to differentiation and sloughing of normal colonies inder these conditions. Using the clonal density Ca++ shift assay, transformation frequencies have been calculated relative to clonogenic cells plated. During the coming year, the reproducibility of modifications in the assay is being verified using 4 carcinogens which are active in mouse epidermis, and 2 in active structural analogs. The effect of 3 retinoid derivatives in transformation frequency and malignent potential in vivo is being quantitated. The normal and carcinogen altered mouse epidermal clones are being examined for Ca++ dependent changes in potein synthesis in an effort to define molecular markers of normal "differentiation" and of transformation on these cells. Such markers are of particular importance, since reliable markers of transformation in lining epithelial cell have not yet been defined. Analysis of possible markers is being performed by polyacrylamide gel electrophoresis of metabolically labell proteins, obtained from transformed vs non transformed subclones of line 291 which have been incubated in culture medium containing a range of Ca++ concentrations. In collaborative efforts monoclonal and polyclonal antibody to keratins and spectrin are being used to assess whether these proteins respond to Ca++ in the subclones. Long term objectives are to use the clonal density epidermal transformation assay to evaluate mechanisms of action of initiating and promoting agents in chemical carcinogenesis, and of "anti tumor" and differentiation modulating drugs in the inhibition of carcinogenesis.